Super-resolution microscopic imaging technique has a significant influence in biological imaging, material characterization, laser fine processing, etc. Among these, stimulated emission depletion (STED) microscope can realize super-resolution imaging by reducing the area of signal using a depletion beam, based on a conventional confocal microscope. Compared with other super-resolution imaging microscopies, STED microscope has a simple principle and can achieve super-resolution imaging of living objects in vivo, with fast imaging speed. STED super-resolution technique provides a new feasible method in biomedical research, nano-materials research, laser fabrication, optical storage, etc.
In conventional STED microscopy, there are two laser beams. One is an unmodulated Gaussian beam, called excitation beam. It can generate a nearly Airy spot after the focus by the objective lens, which can produce fluorescent signals by a fluorescent material at the focus area. The other beam, called depletion beam, is a Gaussian beam modulated by 0-2π vortex phase-plate. It can generate a doughnut shaped focal spot with a nearly zero intensity in the center. In the ring area of high depletion intensity, the excited molecules jump back to the ground state via stimulated emission instead of spontaneous radiation, so only the molecules in the dark center of depletion focal spot generate spontaneous radiation signals. Thus, the depletion beam can help obtain a super-resolution optical imaging. However, the shape of focal depletion spot is sensitive to the phase change. For specimens with large image depths, the resolution can rapidly decrease deep in the specimens because the shape of focal depletion spot is distorted due to spherical aberration, scattering distortion and loss.
In recent years, to improve deep imaging resolution in STED, some researchers achieved stable resolution at 80-100 micron image depth by adjusting a correction collar lens, but this method involves complex operations and is not suitable for high scattering samples. Some other efforts compensate spherical aberration with self-adaptive optics system, but these methods require complex operations or expensive optical system.